This invention relates to a process for separating a complex of a trace component in a sample derived from a living body, for example, a body fluid such as serum, blood, plasma or urine, lymphocyte, hemocyte or any of various cells and a substance having a specific affinity for the trace component, from other substances, and a process for measuring the trace component by utilizing said separating process.
It is known that specific substances interact strongly on each other to form a stable complex. The specific substances include, for example, the following combinations: antigen and antibody; protease and its protein protease inhibitor; sugar chain and lectin; enzyme and substrate therefor or coenzyme; physiologically active substance such as hormone, and receptor or transport protein for said active substance; and a pair of polynucleotide chain of duplex DNA.
As processes for measuring a trace component in a sample by utilizing the above interaction, there are the processes previously developed by the present inventors which have been disclosed in Japanese Patent Unexamined Publications Nos. 2-28557, 3-206964 and 3-221865 (EP 0357869-A and EP 0441470-A).
The outline of the process disclosed in Japanese Patent Unexamined Publication No. 2-28557 (EP 357869-A) is given below by taking the case where the interaction between an antigen and an antibody is utilized.
(1) A process for measuring the amount of an analyte in a sample derived from a living body which comprises mixing the sample containing an analyte to be measured with an anti-analyte antibody, which antibody has been labeled with a substance detectable by some method (hereinafter abbreviated as "detectable substance") (labeled antibody); reacting the analyte with the labeled antibody; separating the resulting complex of the analyte (the antigen) and the labeled antibody from free labeled antibody by a high pressure liquid chromatography (HPLC); and measuring the amount of the detectable substance in the complex.
(2) A process for measuring an analyte in a sample derived from a living body which comprises mixing the sample containing an analyte to be measured with the analyte which has a detectable substance attached thereto and an anti-analyte antibody to be measured; reacting the unlabeled analyte and the labeled analyte with the antibody; separating the resulting complex of the analyte (the antigen) labeled with the detectable substance and the antibody from free analyte (antigen) having the detectable substance attached thereto, by a high pressure liquid chromatography (HPLC); and measuring the amount of the detectable substance in the complex or the amount of the detectable substance in the free analyte (antigen) having the detectable substance attached thereto.
The invention disclosed in Japanese Patent Unexamined Publication No. 3-206964 (EP 0441470-A) is a process for measuring "two or more analytes to be measured having the same action and the same detectable chemical characteristics" such as isozymes, hormones different in sugar chain structure, etc.
This process is explained below by taking the case where saliva .alpha.-amylase and pancreas .alpha.-amylase are analytes to be measured.
This measuring process comprises mixing anti-saliva .alpha.-amylase (mouse) monoclonal antibody with a sample containing the above two .alpha.-amylases to react the same with saliva .alpha.-amylase, separating the resulting complex of saliva .alpha.-amylase and anti-saliva .alpha.-amylase (mouse) monoclonal antibody from free pancreas .alpha.-amylase by HPLC, measuring the amount of saliva .alpha.-amylase contained in the complex of saliva .alpha.-amylase and anti-saliva .alpha.-amylase (mouse) monoclonal antibody and/or the amount of the free pancreas .alpha.-amylase, and thereby separating and measuring saliva .alpha.-amylase and/or pancreas .alpha.-amylase in the sample.
The invention disclosed in Japanese Patent Unexamined Publication No. 3-221865 (EP 0441470-A) is a process for measuring "two or more analytes to be measured having the same action" such as isozymes and hormones different in sugar chain structure, or "two or more analytes to be measured having different actions in spite of their similar structures" such as steroid hormones, human chorionic gonadotropin.
This process is explained below by taking the case where hCG derived from placental villi and hCG derived from choriocarcinoma are analytes to be measured.
This measuring process comprises mixing a sample containing the above two hCG's with anti-hCG-.beta. chain monoclonal antibody having affinity for both hCG's which has been labeled with a detectable substance, and a lectin which has affinity only for hCG derived from choriocarcinoma and does not bind to hCG derived from placental villi; reacting the hCG's with the labeled monoclonal antibody and the lectin; separating the resulting complex of hCG derived from placental villi and the anti-hCG-.beta. chain monoclonal antibody labeled with the detectable substance, the resulting complex of hCG derived from choriocarcinoma, the anti-hCG-.beta. chain monoclonal antibody labeled with the detectable substance and the lectin, and free anti-hCG-.beta. chain monoclonal antibody labeled with the detectable substance, from one another by HPLC; measuring the amount of the detectable substance in each complex; and thereby measuring the amounts of the two hCG's in the sample.
As is clear from the above, the measuring processes disclosed in the above references are characterized in that a complex (or a complex labeled with a detectable substance) formed by the interaction between an analyte to be measured (or an analyte to be measured which has been labeled with the detectable substance) and a substance having affinity therefor (hereinafter abbreviated as "affinity substance") is separated from free affinity substance (or free analyte labeled with the detectable substance) by using HPLC. These processes make it possible to determine the amount of a trace component more easily in a shorter time with much higher precision as compared with conventional measuring processes according to EIA (enzyme immunoassay), RIA (radioimmunoassay), FIA (fluoroimmunoassay) or the like. Therefore, they are thought to be measuring processes of great promise.
In addition, the above references disclose, for example, a process using two or more affinity substances (specifically, two or more affinity substances capable of binding to different sites, respectively, on an analyte to be measured) in the formation of a complex; a process using two or more affinity substances labeled with a detectable substance; the fact that employment of two or more of such affinity substances results in an increased molecular weight of the complex, a higher degree of change of the isoelectric point of the complex, etc. and hence further facilitates the separation of the complex from free affinity substances, so that the precision of measurement can be improved; and the fact that the measuring sensitivity can be increased by labeling each affinity substance with a detectable substance previously.
However, the processes using the two affinity substances (including labeled affinity substances) cannot be utilized when an analyte to be measured has only one site to which the affinity substances can bind. Said processes can cause the phenomenon that properties (molecular weight, hydrophobicity, ionicity, etc.) of the complex are consequently changed, resulting in a shifted position of elution of the complex. But they have been unable to control the above properties of the complex freely.
Therefore, when the two affinity substances are merely used in the separation using HPLC in the above-mentioned processes, the following problems, for example, are caused in some cases. The separation of the complex (or the complex labeled with a detectable substance) from free affinity substance (or free analyte labeled with the detectable substance) is not sufficient, or the position of elution of the complex (or the complex labeled with the detectable substance) is the same as that of a living body component in serum or urine, so that the precision of measurement is lowered. Accordingly, there has been a desire to seek further improvement in said processes.